Apparatus for assaying enzyme activity

ABSTRACT

IMPROVED METHOD AND APPARATUS FOR QUANTITATING THE ACTIVITIES OF A WIDE SELECTION OF ENZYMES PRESENT IN BIO: LOGICAL FLUIDS, USING A SPOT TEST TECHNIQUE. THE TEST ULTILIZES ENZYME STANDARDS FREEZE-DRYED ON TRANSPARENT MEMBRANES ASSEMBLED TOGETHER WITH BUT SEPARATED FROM REAGENTS, INCLUDING SUBSTRATES, DYES, AND COFACTORS, WHICH ARE FREEZEDRYED IN ABSORBENT PADS. THE ASSEMBLY MAY UTILIZED MICROPOROUS MEMBRANES AS FILTERS TO ASSIST IN PROCESSING WHOLE BLOOD SAMPLES. AS ASSEMBLED, THE SPOT TEST PLATES OR SLIDES ARE COMPLETELY SELT-SUFFIIENT, REQUIRE NO ADDED CONTROLS, INSTRUMENTATION, OR LIQUID VOLUME MEASUREMENT. THE TESTS MAY BE UTILIZED UNDER ANY ENVIRONMENTAL CONDITIONS SUCH AS THOSE REQUIRING RAPID DIAGNOSTIC SUPPORT UNDER EMERGENCY CONDITONS, OR ROUTINELY BY MEDICAL PRACTITIONERS IN OFFICES, HOSPITIALS OR CLINICS. TEST PLATES MAY ASSIST IN THE DIAGNOSIS OF A WIDE SPECTRUM OF PATHOLOGICAL CONDITIONS RELATING TO MALFUNCTIONS OF THE LIVER, SPLEEN, HEART, NEURALOGICAL SYSTEM,, LUNGS, KIDNEYS, MUSCLE TISSUE, BONES, ETC.

Jan. l, WM R. H. MOYER ET AL- APPARATUS {"OR ASSAYING ENZYME ACTIVITY Filed Jan. 27, 1971 ENQWMMMM/MV MSWMMMMM AWMMWM '5"WMMM MMIV" ENVEENTDWS RUDOLPH H. MOYER DONALD J. SIBBETT www m @Ww ATTORNEY vUnited States Patent() "ice 3,783,105

Patented Jan. 1, 1974 3,783,105 Hepatobiliary diseases APPARATUS FOR ASSAYING ENZYME ACTIVITY Rudolph H. Moyer, West Covina, and Donald J Sibbett, In application of blood or serum enzyme activity tests Cucamonga, Calif assignmto Geom, Incorporated, to the diagnosis of disorders of the liver and its duct sys- Rockville, Md. tems, it is possible only in relatively few cases to dis- I3 `iled Jan. 27, 1971, Ser. No. 110,185 5 criminate between the possible sources of the enzymatic The portion of the term of the patent subsequent to inbalance indicative of specic pathological conditions.

May 161s 1t9is ilbsi/djiciaimed However, the following enzymes are considered particu- U s Cl 195 ,127 n 11 Claims larly useful in support of diagnosis of such diseases: alkaline hos h ta e lactic deh dro nase lutamic lacetic P P a s Y ge ,g X3

transaminase, isocitric dehydrogenase, sorbitol dehydro- ABSTRACT OF THE DISCLOSURE genase, leucine aminopetidase, aldolase, and 5nucleo- Improved method and apparatus for quantitating the iidase among Othersactivities of a wide selection of enzymes present in bio- Tilo foiiowiiig table shows soms of tho diseases which logical iluids, using a spot test technique. The test ultilizes slssoiiioaiiy give riso io considerably increased ioVois of enzyme standards freeze-dryed on transparent membranes l5 these eiiZYms and the disorders indicated. assembled together with but separated from reagents,

including substrates, dyes, and cofactors, which are freezefggl' giii'dc' dryed in absorbent pads. The assembly may utilize micro- Enzymes jaundice jaundice others porous membranes as filters to assist in processing whole Alkaline phosphatase No Yes Bone dsease blOOd Samples. AS aSSembled, the Spot teSt plates 01 SlideS 20 Lactic dehydrogenases.. Yes 1.... No Myocardial interets,z are `completely self-suicient, require no added controls, iuigrrgiigy" instrumentation, or li uid volume measurement. The tests Glutamic oxolacetic Yes-.-.. Nom- Myocardial interets,

q transarninase pancreatitis may be utilized` under any environmental conditions such Glutamjpmvic Yes No Myoeardalinmts, as those requiring rapid diagnostic support under emer- I treisarrdmkilas Y N Apatncreetitis. l gency conditions, or routinely by medical practitioners in som ne e y ogenase asm" o frardia offices, hospitals or clinics. Test plates may assist in the glbiitilgehydrogenaso- Ige 1go C fth diagnosis of a Wide spectrum of pathological conditions peaino 0 es" amaro epancreas relating to malfunctions of the liver, spleen, heart, neuralglcalg-S Muscular dystrophy. logical system, lungs, kidneys, muscle tissue, bones, etc.

1 Not heat stable. Heat stable. The present invention relates to extensions and improve- Serum and mme' ments of the concepts and principles disclosed in our co- Disease 0f the lung pending application, Ser. No. 63,842, entitled Method and Apparatus for Quaniiiatmg Enzyme Activity iiied Aug' 35 levels in various pulmonary disorders. The Ifollowing table 14,1970iioWPai 3,663,374 indicates briey a number of possibilities. Compared to BACKGROUND cardiovascular and hepatobiliary diseases, elevation of enzyme activity is usually less dramatic in lung diseases. When sharply elevated it is frequently an indicator of A number of experts have reported elevated enzyme Rapid assays of the activities of enzymes in body fluids oder to the practicing physician a tool for conrmation of 40 complications.

Diseases l Pulmonary infarct or Enzymes embolism Pulmonary insuiiiciencies Lung cancer Others Laetic dehydrogenases Positive.-. Positive for some disorders..- Usually positive Myocardial interets, hepatobiliary diseases y other carcinomas. Glutemi'c-oxalacetic transamina e dn d0 Cardjiovastular diseases, liepatobiliary disor ers, e c. .Aldolase Positive Other carcinomas. Phosphohexose isomerase. .do Do. Malc dehydrogenase Frequently elevated... D0. Isocitrio dehydrogenase do l Do.

diagnoses which has been generally unavailable in situa- Pancreatitis tions devoid of trained personnel and suitable instrumentation. In addition, sufficient data on the correspondence between various diseases and enzyme activity variations are not available, partially because only time-consuming or diicult enzyme assay methods are available. Our previous, above-identified patent application, discloses methods for very rapid estimation of enzymes which are The laboratory diagnosis of pancreatitis may be associated with (1) abnormalities in enzyme digestive capacity, and (2) changes in blood and urine enzyme levels. Only amylase and lipase have had extensive clinical trials. Diagnostic possibilities are indicated in the following table.

indicative of cardiovascular disorders, particularly myo- Diseases odjed r Acute Chronic ca'rdlai mfarctlons' Th.ese.meth0ds may be m f-0 Enzymes pancreatitis pancreatitis Others screening enzymes indicative of a wide range of physios, hug sim- Alpha Positive With Usually Adrenocorticel stress logical malfunctions even at subclinical level T ,Y n amylase. serum' higher positive. hepatocellular ple rapid enzyme assays employing the easily available levelsin mine. diseaesygastrointestb body iluids may be conveniently utilized in the practice gscliriiosysiirum) of preventative medicine as well as in assisting in diagosis Lime Usually positive" Negative Drug use, gastroin.'

of pathological conditions. Exemplary examples are set forth hereinbelow.

Diseases Enzymes Neural and Glutamic-oxalacetic transaminase, glutamicmuscular. pyruvic transaminase, creatine phosphokinase,

lactic dehydrogenase, isocitric dehydrogenase, aldolase, cholinesterase, leucine aminopeptidase. Skeletal muscle-.- Aldolase, creatine phosphokinase, lactic dehydrogenase, transaminases, 6-nncleotidase, ATP-ase, phosphohexosisomerase, adplahydrosy butyrlc dehydrogenase. Bone Alkaline phosphatase, acid phosphatase l Urinary, includ- Acid phosphatase, lactic dehydrogenase, alkaline ing prostate phosphatase, glutamic-oxalaeetie transaminase, and kidney. beta-glucuronidase.

Gynecological... -phosphoglueonie acid dehydrogenase, beta-glucuronidase.

IEIematological. Glucose-phosphate dehydrogenase,` -phosphogluconic dehydrogenase, pyruvate klnase, glyeeraldehydr-B-phosphate dehydrogenase,

Aldolase, phosphofruetokinase, phosphohexose isomerase, hexokinase, cholinesterase, methemoglobin reductase, glyoxalase, acid phosphatase, glutamicacetic transaminase, pyrophosphatase.

SUMMARY OF THE INVENTION The method and apparatus utilize a simple spot test procedure in which the rate of color development on reference spots, containing pre-standardized increments of enzyme, is compared directly with that on a test spot where the enzyme in any of the several body uids reacts. Direct comparison of the selected lluid enzyme activity with the enzyme activity on the reference spots eliminates need for temperature control and timing of reaction rates. The test measures enzyme activity in the biological uids and requires neither instrumentation nor an operator. It can be employed for rapid screening in emergency situations where clinical test equipment and trained personnel may not be available; however, use in routine situations is also conceived within the scope of applications, Body tiuids such as urine, blood, cerebrospinal uid, serious cavity effusions, gastric juices, and vaginal tluid may serve as the source of enzymes for assay.

An illustrative embodiment of the apparatus and a working embodiment of a practical method will be explained with reference to the accompanying drawings in which:

FIG. 1 is a plan view of a test plate format embodying the concept of the invention;

FIG. 2 is a partial side elevational view showing in assembled relation the various components utilized in a stacked array in the test plate; and

FIG. 3 is a fragmentary exploded view disclosing an appropriate assembly of the components in a stacked test area in a test plate.

In one form of the test, which is illustrative only, a plate format is used. This test may be used to measure the activity of lactic dehydrogenase, pyruvic kinase, hexokinase, sorbitol dehydrogenase, amylase, or many other enzymes-all indicators of simple or complex physiological problems, some of which have been indicated above. All reagents, along with the enzyme standards appropriate to the assay, are freeze-dryed in pads or discs of absorbent material which are held in assemblies similar to those indicated in the illustrative figures.

The reagent systems utilized for the enzyme assays are similar to those routinely employed in standard assays; however, readout is coupled to dye systems which produce developable colors. A typical dye of application is nitro blue tetrazolium which may be coupled to a number of reduction reactions by use of the electron carrier, N-methyl phenazonium methosulfate. Concentrations of reagents in the absorbent pads are adjusted to provide optimum reaction conditions when the freeze-dryed components are reconstituted by wetting the discs. The following examples illustrate the utility of the method.

EXAMPLE 1 Lactic dehydrogenase Lactic (lactate) dehydrogenase catalyzes the reaction:

Lactate--l-NAD Pyruvate-l-NAD H where NAD=nicotinamideadenine dinucleotide and NAD'H is its reduced form. The indicator color is developed by coupling the reduction of nitro blue tetrazolium (NBT) to NAD-H by use of N-methyl phenazonium methosulfate (PMS) as follows:

The following reagents are described in appropriate literature: Henry, R. J. Chiamori, N., Golub, O. I., and Berkman, S., Amer. J. Clin. Path., 34, 381-398 (1960); Honier, G. M., Yott, B., and Lim, J. G., Amer. I. Clin. Path., 51, 287-292 (1969). Concentrations were adjusted on the basis of experience.

Reagents Concentrations (mg/ml.)

Nictinamide adenine dinueleotide (NAD) 0.8

Sodium lactate. 4.

N-methyl phenazonium methosulfate. 0.04 Reagent A Nitro blue tetrazoleum 0.8

Laetate dehydrogenase As required.

Dextran (Dextran, Clinieal-200,000300,000, 3.0 Reagent B.

Nutritional Biochemicals Corp).

All materials were made up in 0.05 M phosphate buffer, pH=7.5. The NAD and sodium lactate components (Reagent A) were adsorbed on the glass fibre pads, the PMS, NBT, LDH and Dextran (Reagent B) were freeze-dryed on the transparent Nuclepore membrane, where color development was most readily followed.

A test plate generally designated 10 for practicing the invention is shown in detail in FIG. 3. It includes a plurality of test spots on areas A, B, C, D, and E. It consists of three slides, 12, 14, and 16, measuring 11A x 3% inches of opaque, white, high-impact polystyrene. Each slide measures 0.040 inch in depth. Each slide contains tive coincident holes, measuring 0.161 inch in diameter. A top glass iibre pad, 18 (Whatman GF/A glass libre paper is a typical material) is 0.250 inch in diameter. Glass fibre pads, 20 and 22, are 0.161 inch in diameter. These two ll hole 24 through slide 14. Reagent A is absorbed on pads 18, 20, and 22. -Pads 18, 20, and 22 are retained between the upper slide, 12, and a porous lter material, 26, such as Acropor AN-200 (Gelman Instrument Company) or cellulose acetate such as Sepraphore III (Gelman lnstrument Company, Cat. No. 51003). The bottom slide, 16, supports an 1i-inch (O.D.) spacer 28 containing a 0.161-inch hole made of 0.015-inch high-impact polystyrene. Across the spacer opening, a transparent lm 30 such as Nuclepore (General Electric Company, S-micron average pore diameter) is sealed. In preparation of the test plates 5 microliters of Reagent B mixture are freeze- ;lryed on the inner surface of the transparent membrane Assembly of test plates requires a technique which will maintain liquid-tight seals between slide 12 and glass ibre pad, 18, and between slide 14 and membrane 26. Accordingly, a number of sealing methods can be utilized to achieve this purpose. Pressure heat seals on the edges, plastic edge binders, eyelets keyed through all plates, and thermal spot seals have been used to maintain the liquid seal integrity.

For use with whole blood, glass fibre pads are consecutively: (l) washed with glacial acetic acid, (2) washed with glass-distilled water, (3) dryed, (4) saturated with 30 microliters of a solution of bovine serum albumin containing 10 mg./ml. and, (5) dryed under vacuum at room temperature in a desiccator.

The glass fibre discs which serve as reagent reservoirs, remove white and red blood cells to prevent clogging of the membrane filter. In practice, the reagents are applied as indicated and slides 14 and 16 are freeze-dryed separately. 'Ihe third slide, 12, is added as a retainer and the slides are sealed together and packaged in a low humidity atmosphereY 5 relative humidity). Spots A, B, and C contain LDH standards representing normal, elevated, and very high enzyme levels respectively. Spot D is a blank from which sodium lactate has been eliminated. Spot E is for assay of LDH and contains all reagents. A reordering of spots can be appropriate.

In conducting a test, the slide is positioned with the dull, glass fibre surfaces uppermost. Water is applied to Spots A, kB, and C; whole blood to Spots D and E. Fluid uptake is controlled by the glass fibre pads; about 30 microliters is absorbed. Extra lluid remains unabsorbed. After application of the samples and water, the slide is inverted and development of blue color is the measure of reaction. After a few minutes, the intensity of color on Spot E is compared to the color development on Spots A, B, and C. Matching gives a direct measure of LDH activity.

EXAMPLE 2 Glucose--phosphate dehydrogenase (G-6-PDH) Glucose--phosphate (G-6-P){nicotinamide adeninedinucleotide phosphate (NADP)- 6phosphogluconate (6-PG) -l-reduced nicotinamide adenine dinucleotide phosphate (NADP-H) The following concentrations have been utilized in the glass libre layers:

Components Concentrations (mg/m1.) NADP 0.8 PMS 0.04 G6P 4.0 Magnesium acetate: 4H2O 0.8 Nitro blue tetrazoleum 0.8

The glass fibre layers were impregnated with 30 microliters. Preparations are made in 0.05 M phosphate buler adjusted to pH=7.4.

The transparent layer on the bottom slide was coated with: Component Concentrations (mg/ml.) Glycose-G-phosphate dehydrogenase As necessary Dextran 3.0

These were also dissolved in phosphate buffer.

The procedure described in Example 1 was carried out to complete preparation of the test devices. Use is practiced in an identical fashion.

6 EXAMPLE 3 Hexakinase Hexokinase is another glycolytic enzyme which may be assayed by techniques similar to those explained above.

The reactions involved are:

hexokinase Adenosine triphosphate (ATP) -lglucose glucose-G-phosphate (G--P) -ladenosine diphosphate (ADP) Components freeze-dryed in the glass fibre layers are:

Components: Concentrations (mg/ml.)

NADP

G-6-P 4.0 Magnesium acetate: llHZO 0.8 IPMS 0.04 NBT 0.8

These components were applied to the glass fibre layers in 30 microliters of a solution prepared in 0.05 phosphate buffer, adjusted to a pH=7.4.

The transparent layer contained:

Components: Concentrations (mg/ml.) Hexokinase As necessary. G--PD'H 1 I U. Dextran 3.0.

These were also prepared in phosphate buffer and 5 microliters of solution was dryed on each site.

Techniques described in Example l were utilized to prepare and use the test slides.

EXAMPLE 4 Aldolase Fmctose-1,6diphosphate (F1-6 DP) a D-glyceraldehyde 3-phsophate(G-3-P) dihydroxy acetone phosphate (DHAP) G-3 PDH Components freeze-dryed in the glass fibre layers are:

Comp onent: Concentrations (mg/ml.) NAD 0.8 F1-6 DP 4.0

PMS 0.04

NBT 0.8 Disodium arsenate 4.0

This solution is prepared in 0.05 tris buer, pH=7.4. A volume of 30 microliters is used to impregnate the glass bre pads.

The transparent lower layer is coated with microliters of:

Component: Concentration (mg/ml.)

G-3 PDH 2 I.U. Aldolase For standards, as required. Cysteine-hydrochloride 2.0. Dextran 3.0.

The solution is also prepared in 0.5 M tris buffer.

Completion of the preparation and use of these plates is described in Example No. 1.

EXAMPLE 5 Glyceraldehyde-3-phosphate dehydrogenase Components: Concentration (mg/ml.) NAD 0.8 F1-6 DP 4.0 PMS 0.04 NBT 0.8 Disodium arsenate 4.0

This solution is prepared in tris buffer as in Example 4 and used to impregnate the glass libre layers.

The coating on the transparent surface is comprised of Components: Concentration (mg/ml.)

Aldolase 3 I.U. G-3-PDH For standards, as required. Gysteine hydrochloride 2.0. Dextran 3.0.

Most kinase and dehydrogenase enzymes may be assayed by procedures similar to those indicated above. Included among such are: glycerol dehydrogenase, pyruvic kinase, malate dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, alpha-hydroxy butyrate dehydrogenase, glutamic dehydrogenase, sorbital dehydro genase, -phosphogluoonic acid dehydrogenase. With appropriate reaction modifications, assay of alkaline and acid phosphatases and amylase may also be carried out in the indicated format.

Although the foregoing example of test plates have been evolved with nitro blue tetrazoleum as the indicator of enzyme activity, any chromogen may be used which demonstrates an appropriate color change on reduction. Another tetrazoleum dye which is directly applicable is MTI [I5-(4,5 dimethylthiazol-Z)-2,5diphenyltetrazoleum bromide] which is available from G. T. Gurr, Ltd. Dyes which decolorize may also be used, whether singly or in combinations. Such techniques may be utilized to give many color combinations in the test plates. For example, decolorization of 2,6-dichlorophenol-indophenol (a deep blue dye) may be used in conjunction with a permanent yellow dye such as p-amino azobenzene (at basic or neutral pHs) to form an indicator which passes from bluegray to green and eventually becomes yellow. Such color combinations may greatly improve the ease of comparison of the standards and the test spots. This indicator technique with mixed dyes may be used with a wide variety of enzyme reactions.

Manifestly minor changes and variations can be effected in the embodiment shown and described without departing from the spirit and scope of the invention as defined in and limited solely by the appended claims.

We claim:

1. In a system for visually quantitating enzyme activity in biological and the like liuids:

(A) a support;

(B) means constituting a plurality of separate restricted test zone areas in a rigid array mounted on said support;

(C) at least one of said areas including as a stacked array a plurality of superposed test reagent impregnated members in a rigidly confined column and adapted for placement thereon of a fluid test media, said stacked array including in descending sequence:

(i) a top porous glass fiber disc;

(ii) an intermediate porous glass fiber disc; (iii) a lower porous glass fiber disc;

(iv) a porous filter membrane; and

(v) a transparent lilm membrane.

(D) said glass iber discs and said porous filter membrane consistitutng llters to remove contaminants including white and red blood cells;

(E) said glass fiber discs constituting dried reagent storage reservoirs for a iirst test reagent for elution therefrom by clear filtrate passing through the said discs;

(F) said transparent ilm membrane having a second, freeze dried, reagent on the upper surface thereof;

(G) said glass iiber discs constituting fluid control units and functioning to draw liquid therethrough;

(H) said support including first, second and third suerimposed slides having a plurality of sets of mating holes therethrough, a said stacked array being mounted at each of said set of mating holes and confined between said slides.

2. In a system as claimed in claim 1, said top glass fiber disc being contained between said first and second slides and being of a size greater than that of said holes, said intermediate and lower glass fiber discs being closely contained and mounted within the hole in said second slide, said porous filter membrane and said transparent lm membrane being of greater size than said holes and mounted between sair second and third slides.

3. In a system as claimed in claim 2, further including a spacer having a hole mating with said holes of said slides, said spacer interposed between said transparent film membrane and said third slide.

4. In a system as claimed in claim 3, said slides as assembled in an array being sealed together and including liquid tight seals between said first slide and said top porous glass liber disc, and between said third slide and said transparent lm membrane.

5. In a system as claimed in claim 2, wherein some of said stacked arrays are standardized with the enzyme to be assayed, each of the arrays having the enzyme freeze dried on said lilm membrane and separate from said liber discs.

6. In a system as claimed in claim 5, wherein said glass fiber discs contain reagents including substrates, dyes, and co-factors which are freeze-dried therein.

7. In a system as claimed in claim 6, said reagents including for a color readout test result a dye material which indicates activity of the assayed enzyme.

8. In a system as claimed in claim 7, wherein the dye consists of a nitro blue tetrazolium and N-methyl phenazonium methosulfate as an electron carrier to produce a reduction reaction.

9. In a system as claimed in claim 4, wherein five test zone areas are provided, three prearranged said areas containing lactate dehydrogenase standards representing normal, elevated, and very high enzyme levels respectively, a fourth said area constituting a blank and the remaining said area being for lactate dehydrogenase assay of a test specimen and containing all reagents for said assay, said lactate dehydrogenase standards areas being adapted for application thereto of water, and said fourth and remaining area being adapted for application thereto of the test specimen whereby, after such application the assembly is inverted and color develops in the test areas indicative of the measure of reaction and the intensity of color developed in said remaining area, upon matching comparison with color development in the standards containing Reagents: Concentrations Nicotinamide adenine dinucleotide (NAD) 0.8 Sodium lactate 4.0

N-methyl phenazonium methosulfate (PMS) |0.04 Nitro blue tetrazolium (NBT) 0.08 Lactate dehydrogenase (LDH) As required Dextran (Dextran, Clinical200,000-300,000

Nutritional Biochemicals Corp.) 3.0

and wherein, all materials being made up in 0.05 phosphate buffer, pH=7.5, the NAD and sodium lactate com- 10 ponents being adsorbed on said glass bre discs, the PMS, NBT, LDH and Dextran being freeze-dried on the upper surface of the transparent membrane, where color development is most readily followed.

References Cited UNITED STATES PATENTS 3,526,480 9/1970 Findl et al 195-103.5 R 3,367,841 2/1968 yBuissiere et al. 195103.5 R 3,663,374 5/1972 Moyer et al. 195-103.5 R

ALVIN F. TANENHOLTZ, Primary Examiner MAX D. HENSLEY, Assistant Examiner U.S. C1. X.R.

23-253 TP; 195-103.5 R, 100 

